Reactive oxygen species (ROS) and oxidative stress are thought to play a central role in potentiating macrophage activation, causing excessive inflammation, tissue damage, and sepsis. Recently, we have shown that punicalagin (PUN) exhibits anti‐inflammatory activity in LPS‐stimulated macrophages. However, the potential antioxidant effects of PUN in macrophages remain unclear. Revealing these effects will help understand the mechanism underlying its ability to inhibit excessive macrophage activation. Hemeoxygenase‐1 (HO‐1) exhibits antioxidant activity in macrophages. Therefore, we hypothesized that HO‐1 is a potential target of PUN and tried to reveal its antioxidant mechanism. Here, PUN treatment increased HO‐1 expression together with its upstream mediator nuclear factor‐erythroid 2 p45‐related factor 2 (Nrf2). However, specific inhibition of Nrf2 by brusatol (a specific Nrf2 inhibitor) dramatically blocked PUN‐induced HO‐1 expression. Previous research has demonstrated that the PI3K/Akt pathway plays a critical role in modulating Nrf2/HO‐1 protein expression as an upstream signaling molecule. Here, LY294002, a specific PI3K/Akt inhibitor, suppressed PUN‐induced HO‐1 expression and led to ROS accumulation in macrophages. Furthermore, PUN inhibited LPS‐induced oxidative stress in macrophages by reducing ROS and NO generation and increasing superoxide dismutase (SOD) 1 mRNA expression. These findings provide new perspectives for novel therapeutic approaches using antioxidant medicines and compounds against oxidative stress and excessive inflammatory diseases including tissue damage, sepsis, and endotoxemic shock.

Caffeic acid possesses multiple biological effects, such as antibacterial, antioxidant, antiinflammatory, and anticancer growth; however, what effects it has on bovine mastitis have not been investigated. The aim of this study was to verify the antiinflammatory properties of caffeic acid on the inflammatory response of primary bovine mammary epithelial cells (bMEC) induced by lipopolysaccharide (LPS), and to clarify the possible underlying mechanism. Bovine mammary epithelial cells were treated with various concentrations (10, 50, 100, and 200 μg/mL) of LPS for 3, 6, 12, and 18 h; the results showed that LPS significantly inhibited cell viability in a time- and dose-dependent manner. When cells were treated with LPS (50 μg/mL) for 12h, the cell membrane permeability significantly increased, which promoted cell apoptosis. Various concentrations (10, 25, and 50 μg/mL) of caffeic acid could weaken the inflammation injury of bMEC induced by LPS without cytotoxicity. Proinflammatory cytokines (IL-8, IL-1β, IL-6, and tumor necrosis factor α) from bMEC were decreased. Nuclear transcription factor κB activity was weakened via blocking κB inhibitor α degradation and p65 phosphorylation. All these showed that the protective effect of caffeic acid on LPS-induced inflammation injury in bMEC was at least partly achieved by the decreased production of proinflammatory cytokines mediated by the effect of reducing the κB inhibitor α degradation and p65 phosphorylation in the nuclear transcription factor κB pathway. The use of caffeic acid would be beneficial in dairy cows during Escherichia coli mastitis as a safe and natural antiinflammatory drug.

Heat stress (HS) and secondary restricted blood flow to the intestines cause dysfunction of the intestinal epithelial barrier. Tight junctions (TJs) are essential to maintain intestinal integrity. l-Arginine has beneficial effects on gut functions. However, the underlying mechanisms remain largely unknown. This study tested the hypothesis that l-arginine regulates the TJ network by activating AMP-activated protein kinase (AMPK) signaling, which in turn improves intestinal barrier functions under HS. IEC-6 cells and rat small intestines were used as experiment models of heat stress. AICAR and dorsomorphin were used to activate and inhibit the AMPK pathway, respectively. Cell proliferation, apoptosis, differential gene expression and KEGG pathway analysis, intestinal paracellular permeability, intestinal morphology, and expression of HSP and TJ proteins, and p-AMPK were determined. l-Arginine promoted cell proliferation and reduced apoptosis after heat exposure at an optimal concentration of 5 mmol. Transcriptome sequencing analysis revealed that differentially expressed genes associated with the HSP family and TJs were elevated by l-arginine. According to KEGG pathway analysis, l-arginine activated the AMPK signaling pathway. In vivo, intestinal damage resulted in obvious morphological changes as well as apoptosis with TUNEL and caspase-3 staining under HS and dorsomorphin treatments. Furthermore, HS and dorsomorphin increased the serum D-lactate concentration, diamine oxidase activity, and mRNA expression level of MLCK (P < 0.05). In contrast, l-arginine and AICAR treatments reduced intestinal injury, maintained intestinal permeability, and increased the villus/crypt ratio under hyperthermia. l-Arginine had the same effect as AICAR both in vitro and in vivo, namely increasing p-AMPK protein expression. l-Arginine and AICAR also upregulated the mRNA expression level of HSP70 and HSP90, and downregulated mRNA expression of MLCK (P < 0.05). The protein expression levels of TJ proteins ZO-1 and claudin-1 were suppressed by heat stroke and dorsomorphin, but enhanced by l-arginine and AICAR. Our findings indicate that activation of AMPK signaling by l-arginine is associated with improved intestinal mucosal barrier functions by enhancing the expression of TJs in rat small intestines and IEC-6 cells during HS.

The intestinal epithelium plays a critical role in absorbing nutrients and maintaining the integrity of the gut barrier. Extreme heat stress induces damage to the intestinal epithelium. However, the protein expression changes and the mechanism behind this damage remain poorly understood. In this study, morphological observation showed that heat stress induced desquamation of intestinal epithelial cells, and destruction of intestinal microvilli and mitochondria. Heat stress-induced changes in the intestinal proteome were quantified using the iTRAQ method followed by mass spectrometry and software analysis. A total of 1689 proteins were identified in rat intestine tissue, of which 41 showed significantly altered expression between the heat stressed and control groups. However, these proteins with significant alterations were involved in biological processes such as cellular assembly and organization, developmental disorder, organismal injury and abnormalities, and inflammation. We found that members of the MAPK and NF-κB signaling pathways act as hub proteins in the network interaction analysis. Furthermore, western blot analysis verified that the MAPK and NF-κB signaling pathways were activated by heat stress as expected. This study suggests that heat stress induces cell cytoskeleton reorganization and an inflammatory response, and the activation of the MAPK and NF-κB signaling pathways, which may ultimately contribute to intestinal injury.